Abstract: Streamlined Structures of Human Integral Membrane Proteins at Atomic Resolution About 35% of the human genome encodes integral membrane proteins (IMPs) and one-third of approved drugs target this class. While human IMPs are amenable to straightforward biochemistry, structural studies are nearly intractable as evidenced by the paucity of human IMP structures available. To date, only five human IMP structures have been solved to truly atomic resolution (< 3.0 [unreadable]). None of the human IMP structures represent transporters, likely due to the extreme flexibility from alternating access mechanisms impeding crystallography. Sharp resolution of IMPs in multiple conformations is a prerequisite for understanding the full mechanism of transport proteins, the role of amino acids in substrate recognition, drug binding, inter-domain communication and for accurate structure-based drug design. Structure-determination of human IMPs lags decades behind the determination of soluble protein structures. We plan to accelerate the process and simultaneously provide human IMP structures in multiple conformations at atomic resolution. Our innovative strategy utilizes the screening of a synthetic antibody library to rapidly identify high- affinity Fabs (SynFabs) that will serve as scaffolds for crystallography. The versatile SynFab library can recognize virtually limitless numbers of antigens, will trap human IMPs in multiple conformations and will universalize the structure determination process using molecular replacement methods. We have engineered molecular chaperones to enable the production of the most difficult human IMPs in folded mature form using a low-cost yeast expression system. Our innovative and comprehensive strategy will accelerate structure determination of human IMPs by years, shedding light on the mechanisms of serious diseases including Cystic Fibrosis (CF), diabetes, cancer, polycystic kidney disease, inflammation, AIDS and multi-drug resistance (MDR). Public Health Relevance: Many diseases are directly caused by a major class of proteins called integral membrane proteins (IMPs). Obtaining three-dimensional structures of human IMPs has been far too costly and time-consuming for effective drug design or the computational prediction of drug absorption, permeation of drug barriers, and multi-drug resistance. We employ a new strategy to determine structures of human IMPs at highthroughput to integrate these computational approaches and accelerate drug discovery.